Mechanism of Adenlylate Kinases

本项目的目的是通过结合遗传、生化、生物有机和生物物理技术来研究腺苷酸激酶(AK)的结构-功能关系。(1)定点突变将用于鉴定基态和过渡态底物结合所涉及的残基。现有的三个模型(来自核磁共振、X射线和理论研究)将受到严格的测试。其目标是获得一款经过修订或全新的车型。(2)分两个阶段评价点突变对AK构象的影响。第一阶段是使用差异蛋白质核磁共振、差异圆二色谱、盐酸胍诱导的变性、热稳定性和计算机图形来评估突变体中可能的结构扰动和残基的潜在结构作用。第二个阶段是使用质子核磁共振来更定量地评估所选突变体的构象扰动。(3)通过使用各种底物类似物的“互补底物诱变”对上述模型进行进一步的检验和提炼。目标是从底物一侧确认酶残基与底物之间的特定相互作用。

The objective of this project is to study the structure-function relationships of the enzyme adenylate kinase (AK) by a combination of genetic, biochemical, bioorganic, and biophysical techniques. (1) Site-directed mutagenesis will be used to identify the residues involved in the binding of substrates in the ground state and in the transition state. The three existing models (derived from NMR, X-ray, and theoretical studies) will be critically tested. The goal is to obtain a revised or a new model. (2) The effects of point mutation on the conformation of AK will be evaluated in two stages. The first stage is to use difference protein NMR, difference circular dichroism, guanidine-HCl-induced denaturation, thermal stability, and computer graphics to assess possible structural perturbations in the mutant and the potential structural roles of the residues. The second stage is to use proton NMR to evaluate conformational perturbations in selected mutants more quantitatively. (3) The above model will be further tested and refined by "complementary substrate mutagenesis" using various substrate analogues. The goal is to confirm the specific interactions between the enzymic residues and the substrates from the substrate side.